Fibrils of the abundant collagen types I and II incorporate monomers of the low abundance fibrillar collagen types V and XI, respectively, which play a role in regulating type I collagen fibrillogenesis in vivo (1,2). Type V collagen helps regulate the size and shape of type I/V heterotypic fibrils (3-5). In some cases of classical Ehlers-Danlos Syndrome (EDS), a heritable connective tissue disorder, mutations in type V collagen genes (6-10) give rise to type I collagen fibrils of abnormal shape and diameter and cause connective tissue fragility, particularly in skin and joints. In chondrodysplasia, defects in a type XI collagen gene give rise to abnormal type II collagen fibrils (11).
Fibrillar collagens are synthesized as procollagen precursors with N- and C-propeptides that are proteolytically processed to yield mature monomers. Type V collagen is widely distributed in vertebrate tissues as an xcex11(V)2xcex12(V) heterotrimer (12,13) that helps regulate the diameters of fibrils of the abundant collagen type I. Previously, mutations in the human COL5A1 and COL5A2 genes, which encode the pro-xcex11(V) and pro-xcex12(V) chains, respectively, have been identified as the underlying defects in cases of the heritable connective tissue disorder classical EDS (Ehlers-Danlos Syndrome) (formerly EDS types I and II, see Reference (Ref.) 76). However, both COL5A1 and COL5A2 have been excluded in some cases of classical type I (EDS I), while a locus has yet to be identified for the hypermobility type of EDS (formerly EDS type III), a condition marked by gross joint laxity, recurrent joint dislocation, and chronic diffuse musculoskeletal pain not attributable to joint involvement.
Another type V collagen is an xcex11(V)xcex12(V)xcex13(V) heterotrimer, isolated primarily from placenta (17,18), but also reported in uterus, skin, and synovial membranes (12,19-21). The xcex11(V)xcex12(V)xcex13(V) heterotrimer has remained poorly characterized but has a lower melting temperature than the xcex11(V)2xcex12(V) heterotrimer and may be incorporated into heterotypic fibrils. Type XI collagen, in the form of an xcex11(XI)xcex12(XI)xcex13(XI) heterotrimer (22), was first characterized as a minor collagen of cartilage. However, findings of type XI chains in noncartilaginous tissues (23), of type V chains in cartilage (24), and of cross-type heterotrimers composed of xcex12(V) and xcex11(XI) chains (25,26) now suggest that type V and type XI chains constitute a single collagen type in which different combinations of chains associate in a tissue-specific manner.
Complete primary structures of the type V/XI procollagen chains pro-xcex11(V), pro-xcex12(V), pro-xcex11(XI), and pro-xcex12(XI) are known (27-35). The pro-xcex13(XI) chain is thought to be an alternatively spliced product of the gene that encodes the pro-xcex11 chain of type II collagen (13, 24). Full-length cDNA sequences have provided not only the inferred primary structure of each chain, but have also provided probes that have allowed fine mapping of the expression domains of cognate mRNAs (27,36-41). Such studies are important, as the low levels of collagen type V/XI chains have limited biochemical and histochemical analyses of expression in developing and adult tissues. Nucleic acid probes have also enabled those studies which established the causal links between defects in type V/XI chains and genetic diseases (6-11).
Of the fibrillar procollagen chains, only the pro-xcex13(V) remains largely uncharacterized at the nucleotide and amino acid level. The xcex13(V) chain exhibits only limited distribution in mammals and is believed to be the least abundant fibrillar (type V/XI) collagen chain. The limited distribution may reflect a more specialized role than those of the other type V/XI chains. It is the only fibrillar (type V/XI) collagen or procollagen chain for which neither complete primary structure nor nucleic acid probes are available. About a third of the amino acid sequence of the major collagenous domain of the xcex13(V) chain was determined by N-terminal sequencing of proteolytic fragments (42). Nevertheless, a true understanding of the nature of mammalian type V/XI collagen and its roles in development, physiology, disease and treatment requires characterization the pro-xcex13(V) and xcex13(V) chains.
The present invention is summarized in that mammalian xcex13(V) polypeptides and variants thereof are disclosed, as are recombinant materials, including genetic constructs, and methods for their production. The invention is further summarized in that polynucleotides that encode the polypeptides and the variants are also disclosed. The invention is still further summarized in that investigative, diagnostic and therapeutic compositions and methods employing the polypeptides, polynucleotides and related materials, such as antibodies, sense- or antisense oligonucleotides and polynucleotides, and the like, are also disclosed. The chromosomal map positions in humans and mice of the polynucleotides that encode the mammalian xcex13(V) polypeptides are also disclosed.
It is an object of the present invention to enable production of large quantities of mammalian xcex13(V) polypeptide chains for research, diagnostic and therapeutic use.
It is an advantage of the present invention that collagen comprising mammalian pro-xcex13(V) or xcex13(V) chains can be synthesized for any such use.
Other objects, features and advantages of the present invention will become apparent upon consideration of the following detailed description taken in conjunction with the accompanying drawings.
Not applicable.
As used herein, a xe2x80x9cmammalian xcex13(V) polypeptidexe2x80x9d refers to a modified or unmodified polypeptide having an amino acid sequence characteristic of those shown in SEQ ID NO:2 and SEQ ID NO:4, or a novel fragment thereof, especially a fragment that is antigenic or has a biological activity. Preferably, a mammalian xcex13(V) polypeptide exhibits at least one biological activity of mammalian xcex13(V) procollagen or collagen. A mammalian xcex13(V) polypeptide can be a mature protein or a larger protein that can include native or non-native amino acid sequences at the N- or C-terminus or both, a propeptide sequence, or other sequence attached to the mature polypeptide sequence. These sequences can include amino acid sequences that assist in purification, detection, or stabilization of the mammalian xcex13(V) polypeptide.
Within the scope of the invention are polypeptides that have at least 80% amino acid identity to that of either SEQ ID NO:2 or SEQ ID NO:4 over its entire length, and more particularly polypeptides having at least 90% identity, or more preferably at least 95% identity, to that of SEQ ID NO:2 or SEQ ID NO:4, when the sequences are aligned to obtain the highest order match using published techniques. Most preferred are polypeptides having between 97 and 99% amino acid identity to that of SEQ ID NO:2 or SEQ ID NO:4. The term xe2x80x9cidentityxe2x80x9d is given its art recognized meaning. Sequence identity can be determined, for example, using the methods disclosed by Devereux et al. (83), incorporated herein by reference in its entirety.
An polypeptide is, e.g., 80% xe2x80x9cidenticalxe2x80x9d if it contains up to 20 amino acid sequence differences, changes or alterations (including substitutions, deletions, or insertions) per each 100 amino acids in reference sequences SEQ ID NO:2 or SEQ ID NO:4. The differences, changes or alterations can be at any position in the amino acid sequence of either polypeptide and can be interspersed as individual changes or contiguous differences.
A xe2x80x9cmammalian xcex13(V) polynucleotidexe2x80x9d refers to a polynucleotide that encodes any mammalian xcex13(V) polypeptide, or a polynucleotide fragment thereof, or a complement of any of the foregoing. A polynucleotide can be modified or unmodified DNA or RNA, whether fully or partially single-stranded or double-stranded or even triple-stranded. A modified polynucleotide can be chemically or enzymatically induced and can include so-called non-standard bases such as inosine. A preferred polynucleotide comprises any sequence that can encode a polypeptide of SEQ ID NO:2 or SEQ ID NO:4, where the number of such polynucleotides is substantial, in view of the well-known degeneracy in the genetic code. In a most preferred embodiment, the polynucleotide comprises a sequence of polypeptide-encoding nucleotides shown in SEQ ID NO:1 (bases 82 to 5298) or SEQ ID NO:3 (bases 87 to 5321), or is a polynucleotide fragment or complement of any of the foregoing.
Within the scope of the invention are polynucleotides that comprise nucleotide sequences having at least 80% identity to that of any of the foregoing over its entire length, and more preferably polynucleotides comprising sequences having at least 90% identity, or more preferably at least 95% identity, to that of SEQ ID NO:1 or SEQ ID NO:3, when the sequences are aligned to obtain the highest order match using published techniques. A polynucleotide sequence is, e.g., 80% identical if it contains up to 20 nucleotide differences, changes or alterations (including substitutions, deletions, or insertions) per each 100 nucleotides in reference sequences SEQ ID NO:1 or SEQ ID NO:3. The differences, changes or alterations can be at any position in the nucleotide sequence of either polynucleotide and can be interspersed as individual changes or contiguous differences.
Identified herein are certain fragments of the mouse and human polypeptides that were not previously known. These include SEQ ID NO:2 between amino acids 1 and 477, SEQ ID NO:2 between amino acids 564 and 663, SEQ ID NO:2 between amino acids 709 and 721, SEQ ID NO:2 between amino acids 758 and 785, SEQ ID NO:2 between amino acids 819 and 923, SEQ ID NO:2 between amino acids 1008 and 1052, SEQ ID NO:2 between amino acids 1086 and 1245, SEQ ID NO:2 between amino acids 1287 and 1310, SEQ ID NO:2 between amino acids 1334 and 1739, SEQ ID NO:4 between amino acids 1 and 478, SEQ ID NO:4 between amino acids 565 and 664, SEQ ID NO:4 between amino acids 710 and 722, SEQ ID NO:4 between amino acids 759 and 786 , SEQ ID NO:4 between amino acids 820 and 924, SEQ ID NO:4 between amino acids 1009 and 1053, SEQ ID NO:4 between amino acids 1087 and 1246, SEQ ID NO:4 between amino acids 1288 and 1311, and SEQ ID NO:4 between amino acids 1335 and 1745. Polypeptides having at least 80% identity to those polypeptide fragments, and preferably having at least 90%, 95%, 97% and 99% identity, are also within the scope of the invention, as are polynucleotides that encode any such polypeptide fragment.
The invention also includes polynucleotides that hybridize to any of the aforementioned polynucleotides under stringent conditions, such as overnight incubation at 42xc2x0 C. in a solution comprising 50% formamide, 5xc3x97SSC (150 mM NaCl, 15 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5xc3x97 Denhardt""s solution, 10% Dextran Sulfate, and 20 micrograms/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1xc3x97SSC at about 65xc2x0 C. Polypeptides encoded by any of the foregoing polynucleotides are also within the scope of the invention.
The polynucleotide can also be a variant of any of the foregoing. A xe2x80x9cvariantxe2x80x9d as the term is used herein, is a polynucleotide that differs from a reference polynucleotide but retains essential properties. Generally, differences are limited so that the sequences of the reference polypeptide or polynucleotide and the variant are closely similar overall and may be identical in part. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide differs in nucleotide sequence from a reference polynucleotide. A variant polynucleotide may or may not encode an amino acid sequence that differs from the amino acid sequence encoded by the reference polynucleotide. Nucleotide changes can, but need not, result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. A variant of a polynucleotide or polypeptide can be a naturally occurring allelic variant, or can be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides may be made by mutagenesis techniques or by direct synthesis or other method.
In keeping with the present invention, exemplary amino acid sequences of mammalian pro-xcex13(V) proteins, precursors of mammalian xcex13(V) proteins, are disclosed, as are cDNA sequences that encode the exemplified human and murine amino acid sequences. Patterns of expression in developing and adult tissues are examined, and the chromosomal locations of the cognate mouse Col5a3 and human COL5A3 genes are mapped. Full length mammalian pro-xcex13(V) cDNA sequences from mice and humans are disclosed in SEQ ID NO:1 and SEQ ID NO:3, respectively. Pro-xcex13(V) chain encoded by the exemplified murine and human nucleic acid sequences are disclosed in SEQ ID NO:2 and SEQ ID NO:4, respectively. The full-length mouse pro-xcex13(V) cDNA and amino acid sequences will be available at GenBank Accession No. AF176645. The full-length human pro-xcex13(V) cDNA and amino acid sequences will be available at GenBank Accession No. AF177941.
The disclosed amino acid sequences have all of the indicia of procollagen chains. Signal peptide cleavage sites, predicted by the method of Nielsen et al. (82), are after amino acid residue 30 (Ala) in the mouse protein and after amino acid residue 29 (Ala) in the human protein. Pro-xcex13(V) is closely related to the xcex11(V) precursor, pro-xcex11(V), but with marked differences in N-propeptide sequences, and collagenous domain features that provide insights into the low melting temperature of xcex11(V)xcex12(V)xcex13(V) heterotrimers, lack of heparin binding by xcex13(V) chains and the possibility that xcex11(V)xcex12(V)xcex13(V) heterotrimers are incorporated into heterotypic fibrils.
In a related aspect, any polynucleotide sequence of the present invention, or an antisense version thereof, can be provided in a vector or genetic construct in a manner known to those skilled in the art. A polypeptide-encoding polynucleotide so provided in a vector can, but need not, be under the transcriptional control of one or more regulatory elements which can include a promoter not natively found adjacent to the polynucleotide such that the encoded polypeptide can be produced when the vector is provided in a compatible host cell or in a cell-free transcription and translation system. Such cell-based and cell-free systems are well known to the skilled artisan. Cells comprising a vector containing a polynucleotide of the invention are themselves within the scope of the invention.
Collagen and derivatives of collagen (gelatin) have been used in medical, pharmaceutical and consumer products for about 40 years. Examples of approved use of collagen include hemostats, vascular sealants, tissue sealants, implant coatings, injectable for plastic surgery, food additives, dental implants, artificial dura, wound dressings, antiadhesion barriers, antibiotic wound dressing, and platelet analyzer reagents. Human and animal collagen can be recombinantly reproduced. The disclosure of the full-length mouse pro-xcex13(V) cDNA and the full-length human pro-xcex13(V) cDNA in the present invention makes it possible to recombinantly reproduce human and animal collagen xcex13(V), which can be used in the applications described above. In addition, human pro-xcex13(V) has been found to express in many tissues including mammary gland, placenta, uterus, brain, fetal lung, and fetal and adult heart. The present invention allows the reproduction of collagen xcex13(V) for the purpose of matching its natural role in the body. Thus, if any of the above tissue is damaged, collagen xcex13(V) can be produced and used in the tissue repairing process.
The polynucleotides of the invention can also be employed as diagnostic reagents in assays for diagnosing a disease or susceptibility to a disease associated with xcex13(V) chains in human or non-human animals. Assays for detecting mutations in protein-encoding sequences are well known to the skilled artisan and can include assaying for changes in primary structure of a fragment by nucleotide sequence analysis, by digesting mismatched hybrids with RNase or by measuring changes in hybrid melting temperatures. Changes in sequence length resulting from insertion or deletion can be observed as a change in electrophoretic mobility of amplified fragments. The present invention also enables other methods for diagnosing changes in an xcex13(V)-encoding polynucleotide, such as nuclease protection assays, for one of ordinary skill in the art. A skilled artisan understands that such assays for diagnosing genetic changes at a fine scale in polynucleotides that encode xcex13(V) chains can be facilitated by providing an array of fragments of the polynucleotides of the invention for systematic screening in parallel for changes at any of a plurality of positions. This methodology enables an association between one or more mutations and a susceptibility to a disease such as classical or hypermobility type of EDS or diseases of other tissues in which xcex13(V) expression is noted such as diseases of female reproductive tissues or the heart as well as various other genetic diseases of the musculoskeletal system, connective tissue or skin.
The present invention also enables one to diagnostically determine whether a human or non-human animal exhibits an altered (e.g., increased or decreased) amount of an xcex13(V) chain or an mRNA that encodes xcex13(V) in one or more tissues of interest. Methods for measuring polynucleotide levels are well known in the art and include quantitative PCR, Northern blotting, dot blotting and others. Methods for measuring protein levels are also known and include ELISA, radioimmunoassay, competitive-binding assays and Western blotting.
Thus, the invention is also embodied in a diagnostic kit comprising one or more of any polynucleotide of the invention, a complementary sequence (antisense) to any polynucleotide of the invention, a polypeptide of the invention, or an antibody or single chain antibody against a polypeptide of the invention or against an immunogenic fragment thereof. An antibody can be obtained in any of several well-known methods such as hybridoma or trioma techniques and can also have utility in purifying xcex13(V) polypeptides or in treating diseases associated with the presence of xcex13(V).
An immunological response effective to protect a human or non-human mammal against undesired activities of wild type or mutant xcex13(V) polypeptides can also be raised in vivo by administering to the mammal an immunogenic polypeptide (either directly or by administering to the mammal a genetic vector comprising sequences that direct expression of the polypeptide under the control of a transcriptional promoter). A vaccine of this type can also include a suitable carrier or adjuvant and can be administered at standard dosages according to standard protocols. The vaccine is preferably administered parenterally by injection, but can also be administered by any route known to be effective for inducing an immune response.
The polypeptides of the invention also enable a skilled artisan to screen for agonists and antagonists of the polypeptides that can be selected using standard screening protocols that include the steps of expressing the polypeptide in or on suitable host cells, exposing the cells to various test compounds, and observing whether any test compound binds to the polypeptide or stimulates or inhibits any biological activity of the polypeptide relative to the binding or activity of the polypeptide in or on untreated control cells. The host cells can be any cells capable of expressing the polypeptide and can include mammalian cells, insect cells, yeast cells, or bacterial cells. Envisioned agonists and antagonists can include, but are not limited to, fragments of the full-length pro-xcex13(V) or xcex13(V) polypeptides that compete biologically with the full-length polypeptides as well as ligands, enzymes, receptors and the like that block active sites on the polypeptides and prevent their interaction with other molecules.
In another aspect, then, the invention extends to a screening kit for identifying agonists or antagonists of the polypeptides of the invention, where the kit contains at least one polypeptide of the invention, an isolated cell or portion of a cell (such as a cell membrane) that contains a polypeptide of the invention, or an antibody to a polypeptide of the invention. In yet another aspect, agonists and antagonists so obtained are within the scope of the invention.
In a therapeutic method, an agonist or antagonist can also be administered along with a pharmaceutically acceptable carrier to enhance or inhibit, respectively, a biological activity of the pro-xcex13(V) or xcex13(V) polypeptides. If the agonist or antagonist is itself a polypeptide or oligopeptide, it can be administered directly (with or without a suitable pharmaceutical carrier) or can be produced in vivo after administration of an expressible genetic vector that encodes the agonist or antagonist or a cell that contains the expressible genetic vector. Alternatively, expression of the pro-xcex13(V) or xcex13(V) polypeptides can be inhibited by administering an antisense sequence of the present invention to interfere with normal polypeptide expression. The antisense sequence can be administered directly (with or without a carrier) or can be produced in vivo after administration of a genetic vector capable of transcribing antisense genetic sequences. Appropriate dosages of an agonist or antagonist will vary depending upon the route of administration and the activity of the administered compound, but can readily be determined and optimized by a skilled artisan. Dosages in the range of between about 0.1 and 100 xcexcg/kg are generally appropriate.